RESUMO
Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
Assuntos
Técnicas Biossensoriais , DNA Bacteriano/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Inibidores da Síntese de Ácido Nucleico/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração Inibidora 50 , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Inibidores da Síntese de Ácido Nucleico/química , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Resposta SOS em Genética/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Naphthyridones that were recently described as a class of translation inhibitors in gram-positive bacteria mediate their mode of action via GyrA in Haemophilus influenzae and Escherichia coli. These are the first examples of compounds in which modes of action in different bacterial pathogens are mediated through widely different targets.